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Survival of the replication checkpoint deficient cells requires MUS81-RAD52 function.

Murfuni I, Basile G, Subramanyam S, Malacaria E, Bignami M, Spies M, Franchitto A, Pichierri P - PLoS Genet. (2013)

Bottom Line: Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate.Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81.However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.

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MUS81 and RAD52 differently affect restart of stalled forks upon CHK1 inhibition.(A) GM01604 cells were transfected with siRNAs directed against GFP (siCtrl), MUS81 (siMUS81) or RAD52 (siRAD52). Replication sites were first labeled with CldU (red signal), left untreated or treated as indicated, followed by recovery for 45 min in IdU (green signal). After immunostaining with antibodies specific for CldU and IdU, the overlapping foci were quantified in each isolated red-positive cell and results expressed as the percentage of CldU/IdU colocalising foci (0–20% of total CldU foci; 20–60% of total CldU foci; 60–100% of total CldU foci). Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. The images shown in the panel (B) are representative of labeling and of different classes of colocalising nuclei.
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pgen-1003910-g005: MUS81 and RAD52 differently affect restart of stalled forks upon CHK1 inhibition.(A) GM01604 cells were transfected with siRNAs directed against GFP (siCtrl), MUS81 (siMUS81) or RAD52 (siRAD52). Replication sites were first labeled with CldU (red signal), left untreated or treated as indicated, followed by recovery for 45 min in IdU (green signal). After immunostaining with antibodies specific for CldU and IdU, the overlapping foci were quantified in each isolated red-positive cell and results expressed as the percentage of CldU/IdU colocalising foci (0–20% of total CldU foci; 20–60% of total CldU foci; 60–100% of total CldU foci). Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. The images shown in the panel (B) are representative of labeling and of different classes of colocalising nuclei.

Mentions: MUS81 has been involved in replication restart after prolonged replication inhibition [25]. Having shown that, in the absence of CHK1 function, RAD52 and MUS81 cooperate in the formation of DSBs at stalled forks, and that their function is required to ensure viability of replication-stressed checkpoint-deficient cells, we studied their relationship with restart of such collapsed forks. Using a double CldU/IdU labelling approach on interphase nuclei, we examined the ability of MUS81 or RAD52-depleted cells to restart replication forks [18]. MUS81 or RAD52 down-regulation did not reduce the number of cells that incorporate the first label (CldU) as compared to the wild-type cells (siCtrl and data not shown), but differently affected incorporation of the second label (IdU) at active replication factories (Figure 5A and B). As expected, CHK1 inhibition severely decreased the ability of HU-treated cells to restart DNA synthesis at stalled forks, as evidenced by the absence of nuclei with more than 60% of CldU/IdU colocalizing foci (Figure 5A and B). A reduction of the ability to incorporate the second label was also observed in MUS81-depleted cells in both unperturbed and HU-exposure conditions (Figure 5A and B). In cells treated with UCN-01 and HU, MUS81 down-regulation did not modify the extent of restart at active replication foci (Figure 5A and B). Interestingly, in RAD52-depleted cells, the increased number of CldU-positive nuclei in which IdU is incorporated at active nuclear foci, demonstrates that HU-stalled forks were recovered in the presence of UCN-01 (Figure 5A and B). Moreover, in UCN-01 and HU-treated cells, RAD52 down-regulation apparently enhanced also the number of nuclei showing only IdU-positive replication foci, which were barely detectable in MUS81-depleted cells (data not shown).


Survival of the replication checkpoint deficient cells requires MUS81-RAD52 function.

Murfuni I, Basile G, Subramanyam S, Malacaria E, Bignami M, Spies M, Franchitto A, Pichierri P - PLoS Genet. (2013)

MUS81 and RAD52 differently affect restart of stalled forks upon CHK1 inhibition.(A) GM01604 cells were transfected with siRNAs directed against GFP (siCtrl), MUS81 (siMUS81) or RAD52 (siRAD52). Replication sites were first labeled with CldU (red signal), left untreated or treated as indicated, followed by recovery for 45 min in IdU (green signal). After immunostaining with antibodies specific for CldU and IdU, the overlapping foci were quantified in each isolated red-positive cell and results expressed as the percentage of CldU/IdU colocalising foci (0–20% of total CldU foci; 20–60% of total CldU foci; 60–100% of total CldU foci). Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. The images shown in the panel (B) are representative of labeling and of different classes of colocalising nuclei.
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Related In: Results  -  Collection

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pgen-1003910-g005: MUS81 and RAD52 differently affect restart of stalled forks upon CHK1 inhibition.(A) GM01604 cells were transfected with siRNAs directed against GFP (siCtrl), MUS81 (siMUS81) or RAD52 (siRAD52). Replication sites were first labeled with CldU (red signal), left untreated or treated as indicated, followed by recovery for 45 min in IdU (green signal). After immunostaining with antibodies specific for CldU and IdU, the overlapping foci were quantified in each isolated red-positive cell and results expressed as the percentage of CldU/IdU colocalising foci (0–20% of total CldU foci; 20–60% of total CldU foci; 60–100% of total CldU foci). Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. The images shown in the panel (B) are representative of labeling and of different classes of colocalising nuclei.
Mentions: MUS81 has been involved in replication restart after prolonged replication inhibition [25]. Having shown that, in the absence of CHK1 function, RAD52 and MUS81 cooperate in the formation of DSBs at stalled forks, and that their function is required to ensure viability of replication-stressed checkpoint-deficient cells, we studied their relationship with restart of such collapsed forks. Using a double CldU/IdU labelling approach on interphase nuclei, we examined the ability of MUS81 or RAD52-depleted cells to restart replication forks [18]. MUS81 or RAD52 down-regulation did not reduce the number of cells that incorporate the first label (CldU) as compared to the wild-type cells (siCtrl and data not shown), but differently affected incorporation of the second label (IdU) at active replication factories (Figure 5A and B). As expected, CHK1 inhibition severely decreased the ability of HU-treated cells to restart DNA synthesis at stalled forks, as evidenced by the absence of nuclei with more than 60% of CldU/IdU colocalizing foci (Figure 5A and B). A reduction of the ability to incorporate the second label was also observed in MUS81-depleted cells in both unperturbed and HU-exposure conditions (Figure 5A and B). In cells treated with UCN-01 and HU, MUS81 down-regulation did not modify the extent of restart at active replication foci (Figure 5A and B). Interestingly, in RAD52-depleted cells, the increased number of CldU-positive nuclei in which IdU is incorporated at active nuclear foci, demonstrates that HU-stalled forks were recovered in the presence of UCN-01 (Figure 5A and B). Moreover, in UCN-01 and HU-treated cells, RAD52 down-regulation apparently enhanced also the number of nuclei showing only IdU-positive replication foci, which were barely detectable in MUS81-depleted cells (data not shown).

Bottom Line: Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate.Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81.However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.

Show MeSH